Structural insights into the Ca2+ and PI(4,5)P2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

Abstract

Proteins containing C2 domains are the sensors for Ca(2+) and PI(4,5)P2 in a myriad of secretory pathways. Here, the use of a free-mounting system has enabled us to capture an intermediate state of Ca(2+) binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P2 and IP3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3-β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P2/PI(3,4,5)P3, whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI(4,5)P2. Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca(2+)-dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.

Keywords: PIP2; calcium; vesicle fusion.

Fig. 1.

Structure of rabphilin 3A–C2A domain bound to Ca²⁺. Ribbon diagrams of the calcium-binding region in the one Ca²⁺ bound (A; yellow) and unbound (B; green) C2A structures. The five critical Asp residues involved in Ca²⁺ coordination are represented by sticks. Coordination interactions are represented by dashed black lines. The initial weighted |Fo|-|Fc| electron density map (contoured at 3.5 σ) calculated in absence of the Ca²⁺ ion is shown as dark blue mesh around the ligand molecule.

Fig. 2.

Three dimensional comparison of C2A domain structures of Ca²⁺-free rabphilin 3A and syt1. (A) Three dimensional overlap of Cα of the C2 domains from rabphilin 3A (PDB ID code 2CHD, gray) and syt1 (PDB ID code 1RSY, green). Sticks represent the side chains of the critical residues involved in the different conformational changes. (B) Similar representation turned 90° to the right.

Fig. 3.

Structure of rabphilin C2A domain bound to PI(4,5)P₂. (A) The C2A–PI(4,5)P₂ complex. The C2A molecule is shown in green with the side chains of amino acids within the β3–β4 groove, directly interacting with the phospholipid depicted as sticks. The phosphate ions and phospholipid molecule are shown as sticks in atom type color. (B, Right) Close-up view of the β3–β4 groove, indicating the interactions between the C2A domain and the PI(4,5)P₂ ligand. The C2A contacting residues and the corresponding ligands are represented in sticks and explicitly labeled. Hydrogen bonds are shown as dashed lines in black. The weighted 2|Fo|-|Fc| electron density map (contoured at 1.5 σ) is shown as blue mesh around the ligand molecule.

Fig. 4.

Binding of C2A (A) and C2B (B) domains of rabphilin 3A assessed by protein-to-membrane FRET. Small unilamellar vesicles (SUVs) composed of POPC/POPS/PI(4,5)P₂/N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (dDHPE) (molar ratio 65:25:5:5) (●,▲), POPC/PI(4,5)P2/dDHPE (molar ratio 90:5:5) (○), and POPC/POPS/dDHPE (molar ratio 70:25:5) (□) were titrated into a solution containing 0.8 µM of C2 domains in the presence of 100 µM CaCl₂ (●) or 1 mM EGTA (▲). A 57% correction was used to calculate the PI(4,5)P₂ or POPS available for C2 domain binding. Effect of mutations K423A and K435A/R437A in the C2A domain (C) and K581A and K593A/K595A in the C2B domain (D) on lipid binding. Data correspond to titrations of SUVs composed of POPC/POPS/PI(4,5)P₂/dDHPE (molar ratio 70:25:5:5) into solutions containing 0.8 µM of wild-type C2 domains (●), C2A (K423A) (Δ), C2A (K435A/R437A) and C2B (K593A/K595A) (○), and C2B (K581A) (▲).

Fig. 5.

Docking model of the C2A domain of rabphilin 3A on the membrane surface. The Ca²⁺-free C2A–PI(4,5)P₂ complex determined in this work was docked on the membrane (A). To simulate the two Ca²⁺-bound forms, we used the PDB ID code 2K3H structure and the PI(4,5)P₂ molecule was added assuming the information obtained in this work (B). The model membrane corresponds to a POPC molecular dynamic simulation with PDB ID code popc128a. Half of the bilayer is represented by thin sticks. Take into account that the C2A–PI(4,5)P₂ complex was docked on the lipid bilayer based on the crystal structure solved in this work. Due to the rotation capacity of the different moieties of the phospholipid, including phosphate 1, the acyl chains might adopt different orientations with respect to the phosphoinositide ring, mainly depending on the restrictions imposed by the membrane dynamic and this might also influence the docking orientation of the domain.

Fig. 6.

Effect of PI(4,5)P₂ on the induction of aggregation of C2A and C2B domains of syt1 and rabphilin 3A. SUVs (0.27 mM) of different composition [POPC/POPS/PI(4,5)P₂, POPC/POPS, EGTA-POPC/POPS/PI(4,5)P₂ for C2A and POPC/POPS/PI(4,5)P₂, POPC/PI(4,5)P₂, POPC/POPS, EGTA-POPC/POPS/PI(4,5)P₂ for C2B and C2A–E194K, from Left to Right in each group of the figure] were incubated with increasing concentration of syt1 C2A (A), C2B (B), or C2A–E194K (C) domains. The same concentrations of SUV of different compositions were incubated with increasing rabphilin 3A: C2A [POPC/POPS/PI(4,5)P₂, POPC/PI(4,5)P₂, POPC/POPS, EGTA-POPC/POPS/PI(4,5)P₂, K435A/R437A-POPC/POPS/PI(4,5)P₂ EGTA-K435A/R437A-POPC/POPS/PI(4,5)P₂ (D)] or C2B [POPC/POPS/PI(4,5)P₂, POPC/PI(4,5)P₂, POPC/POPS, EGTA-POPC/POPS/PI(4,5)P₂, K593A/K595A-POPC/POPS/PI(4,5)P₂, EGTA-K593A/K595A-POPC/POPS/PI(4,5)P₂ (E)] domains. The increment in Abs at 405 nm was measured after 30 min at 25 °C. Cartoons corresponding to the 3D structures of C2A and C2B domains of syt1 (PDB ID codes 1BYN and 1K5W, respectively) and C2A and C2B domains of rabphilin 3A (PDB ID codes 2K3H and 3RPB, respectively) with the bound Ca²⁺ ions shown as yellow spheres and the side chains of critical basic and acidic residues represented as sticks. CBR, lysine-rich cluster (LRC) and bottom face (BF) are also indicated. (F) Model for membrane crosslinking by the C2B domain of rabphilin 3A. The model membranes correspond to a POPC molecular dynamic simulation with PDB code popc128a; they have been oriented to represent a secretory/synaptic vesicle about 40 nm in diameter (Top) and the bended plasma membrane by the simultaneous PI(4,5)P₂/Ca²⁺ interaction as explained in Fig. 5 A and B.